Abstract Although the present treatment options for patients with myelofibrosis (MF) clearly improve their performance status as well as symptoms related to splenomegaly, these approaches has not substantially altered the risk of disease progression or evolution to acute myeloid leukemia. MF arises ultimately at the level of the hematopoietic stem cell (HSC) due to a sequence of genetic events. These malignant MF HSCs and their progeny termed, hematopoietic progenitor cells (HPCs), reside not only within the marrow but also within extra medullary sites such as the spleen. The components of these tissue specific microenvironments act in close proximity to create niches which favor the predominance of malignant HSC/HPC at the expense of normal HSC/HPC. This interplay between the intrinsic properties of MF HSC/HPC and these tissue specific micro- environments results in an inflammatory milieu which promotes MF disease progression. We hypothesize that improvement in the outcomes of MF patients can only be achieved by the implementation of strategies which result in the depletion or actual elimination of MF HSCs and re-establishment of normal hematopoiesis. To accomplish this goal, we hypothesize that the administration of a combination of drugs will be required which would be capable of depleting malignant HSCs as well as disarming the MF HSC promoting microenvironment. Recently, we have demonstrated that MF HSCs are characterized by up-regulation of HDM2 which promotes the proteosomal degradation of p53. Treatment with nutlins, which antagonize the activity of HDM2, up- regulates p53 resulting in the selective depletion of MF HSCs. We recognize that the MF inflammatory milieu is the consequence of up-regulation of NF-?B, a transcription factor which controls pro-inflammatory cytokine production by both myeloid and microenvironmental cells. Increments in p53 levels are also capable of antagonizing NF-?B thereby providing a pharmacological tool with which to blunt the MF inflammatory milieu. We will, therefore, plan to deplete or eliminate MF HSCs by not only up-regulating p53 within MF HSCs, but also within myeloid and microenvironmental cells. Treatment of MF myeloid cells with a nutlin led to the reduction of the production of a number of cytokines including lipocalin-2 which promotes the predominance of MF HSCs over normal HSCs and acts to create an MF like microenvironment. Furthermore we will also target the MF HSC microenvironment by utilizing a CXCR1/CXCR2 (IL-8 receptor) antagonist thereby eliminating actions of IL-8, a chemokine produced in response to lipocalin2 by splenic microenvironmental cells, which promotes the development of MF-HSC promoting vascular niches within the spleen. In addition, we will utilize bromodomain inhibitors to down regulate NF-?B in order to further reduce the elaboration of MF promoting cytokines including IL-8. The combinations of drugs identified by the completion of these planned studies will enact the two pronged strategy outlined above to deplete MF HSCs and will be subsequently evaluated in several paradigm shifting phase1/2 clinical trials in MF patients (Project 4).